Increasing oil consumption makes continued dependence on petroleum reserves untenable. Microbial production of renewable alternatives can reduce petroleum footprints through the in vivo synthesis of ethanol, biodiesel, and industrial precursors (Curran et al. 2013; Elshahed 2010; Li et al. 2008; Xu et al. 2013; Yim et al. 2011). Economic viability is highly dependent upon microbial choice, and an ideal host efficiently generates high titers independent of fermentation condition, through native or imported biosynthetic metabolism (Alper and Stephanopoulos 2009). In this regard, Yarrowia lipolytica's genetic tractability, efficient utilization of many energy sources, and native capacity to accumulate lipids make it an ideal platform for oleo-chemical synthesis (Barth and Gaillardin 1996; Beopoulos et al. 2009a; Papanikolaou and Aggelis 2002).
Here we have employed a large-scale combinatorial approach to maximize lipid production in Y. lipolytica through both genomic engineering and combinatorial and inverse metabolic engineering multiplexed with phenotypic induction.
Y. lipolytica has a fully defined metabolic engineering toolbox that enables intracellular flux control through genomic manipulation (Blazeck et al. 2013b; Dujon et al. 2004; Fickers et al. 2003; Juretzek et al. 2001; Matsuoka et al. 1993). Y. lipolytica is commonly utilized for heterologous protein excretion and to examine and manipulate lipid and fatty acid metabolism (Beopoulos et al. 2009b; Beopoulos et al. 2008; Dulermo and Nicaud 2011; Madzak et al. 2004; Thevenieau et al. 2009), and has proven amenable to downstream manipulation of its fatty acid content to alter desaturation levels (Chuang et al. 2010) or to synthesize novel oleo-chemicals (Blazeck et al. 2013a). Thus, Y. lipolytica lipid reserves are ideal for in vivo catalysis to alkanes (Schirmer et al. 2010), fatty acid esters (Shi et al. 2012) or for standard transesterification-based conversion and use as biodiesel. In particular, biodiesel production grants a high net energy gain compared to other alternative fuels with minimal environmental impact, and harvesting lipid reserves from a microbial source such as Y. lipolytica enables easily scaled-up production without compromising food supply (Christophe et al. 2012; Hill et al. 2006; Kirstine and Galbally 2012; Subramaniam et al. 2010). Y. lipolytica's natural lipid content consists of predominantly C16:0, C16:1, C18:0, C18:1, and C18:2 fatty acids (Beopoulos et al. 2008; Blazeck et al. 2013a; Tai and Stephanopoulos 2013), very similar to the fatty acid content of biodiesel derived from soybeans and rapeseed (Gruzdiene and Anelauskaite 2011; Hammond et al. 2005). Economic viability can be greatly improved by fully utilizing all sugars from lignocellulosic biomass or by using carbon from industrial waste streams. In this regard, Y. lipolytica can efficient utilize hydrophobic and waste carbon sources, such as crude glycerol (Andre et al. 2009; Fickers et al. 2005; Makri et al. 2010; Rywinska et al. 2013), and has shown excellent heterologous gene expression when utilizing glucose, sucrose, glycerol, or oleic acid as a carbon source (Blazeck et al. 2013b). Finally, Y. lipolytica is regarded as a “safe-to-use” organism (Groenewald et al. 2013).
Lipid accumulation in Y. lipolytica can be induced by nitrogen starvation and has been associated with the activity of four enzymes: AMP Deaminase (AMPDp), ATP-Citrate Lyase (ACLp), Malic Enzyme (MAEp) and Acetyl-CoA Carboxylase (ACCp) (Beopoulos et al. 2009a; Dulermo and Nicaud 2011). AMPDp cleaves AMP into NH4+ and inosine 5′-monophosphate to replenish intracellular nitrogen levels; AMP deficiency inhibits the citric acid cycle resulting in citric acid accumulation. ACLp cleaves citric acid into oxaloacetate and acetyl-CoA, and ACCp carboxylates acetyl-CoA into malonyl-CoA fatty acid building blocks. Fatty acid synthesis is further encouraged by a MEAp-mediated increase in NADPH levels (Beopoulos et al. 2009a). Fatty acids can be directly stored in intracellular lipid bodies or further incorporated in triacylglycerides before storage (Beopoulos et al. 2008). Triacylglyceride synthesis follows the Kennedy Pathway to fuse three fatty acids to a glycerol-3-phosphate (G3P) backbone (Kennedy 1961). The ultimate step is catalyzed by the DGA1 or DGA2 acyl-CoA:diacylglycerol acyltransferases (Beopoulos et al. 2009a; Beopoulos et al. 2012). G3P backbone is synthesized from dihydroxyacetone phosphate (DHAP) by the cytosolic, NAD+-dependent glycerol-3-phosphate dehydrogenase (GPD1) and recycled into glycolysis by the mitochondrial, FAD+-dependent glycerol-3-phosphate dehydrogenase isoform (GUT2) (Dulermo and Nicaud 2011). TAG hydrolysis mobilizes free fatty acids for peroxisomal degradation through the four step β-oxidation cycle (Beopoulos et al. 2011)—oxidation by one of six acyl-CoA oxidases (POX1-6), hydration and dehydrogenation by the multifunctional enzyme (MFE1), and thiolysis by a 3-ketoacyl-CoA-thiolase (POT1 or PAT1) (Beopoulos et al. 2009a). The PEX10p transcription factor has been implicated in peroxisomal biogenesis and Δpex10 mutants display increased triacylglyceride content (Blazeck et al. 2013a; Hong et al. 2012; Zhu et al. 2012).
Genomic modifications to Y. lipolytica 's fatty acid, lipid, and central carbon metabolism have shown promise towards increasing lipid accumulation capacity. Deletion of the six POX genes increased ex novo incorporation of oleic acid in Y. lipolytica, while deletion of the single MFE1 gene had a similar effect (Beopoulos et al. 2008; Dulermo and Nicaud 2011). Increasing G3P backbone levels by combining GUT2p deletion and GPD1p overexpression in these β-oxidation deficient backgrounds further increased ex novo lipid accumulation to 65-75% triacylglyceride content (Dulermo and Nicaud 2011). Overexpression of DGA1p increased de novo triacylglyceride accumulation fourfold over control levels to 33.8% triacylglyceride content, and co-overexpression of ACC1p further increased triacylglyceride accumulation to a final yield of 41% triacylglyceride content (Tai and Stephanopoulos 2013). To date, no study has attempted to combine the beneficial effects of engineering Y. lipolytica's fatty acid, lipid and central metabolism in a single strain. Additionally, Y. lipolytica's dependence on media formulation for lipid accumulation has not been adequately explored, nor has its ability to randomly accumulate mutations that enhance lipid accumulation. Furthermore, no attempt has been made to utilize mutation-based evolutionary selection to identify novel lipogenic genotypes. Thus, the ultimate capacity of Y. lipolytica to accumulate lipids and other oleochemicals has not been unlocked. To this end, we have employed a large scale combinatorial approach to maximize lipid production while accounting for unexpected interactions between genotype and environmentally-induced phenotype. The present invention provides solutions to these and other problems in the art.